Chimeric neurotoxins

ABSTRACT

The present invention relates to chimeric neurotoxins with enhanced properties and their use in therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage filing of International PatentApplication No. PCT/EP 2017/060821, filed May 5, 2017, which claims thepriority of United Kingdom Application No. 1607901.4, filed May 5, 2016.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 4, 2018, isnamed SequenceListing16094254.txt and is 550,284 bytes in size.

FIELD OF THE INVENTION

The present invention relates to chimeric neurotoxins with enhancedproperties and their use in therapy.

BACKGROUND

Bacteria in the genus Clostridia produce highly potent and specificprotein toxins, which can poison neurons and other cells to which theyare delivered. Examples of such clostridial toxins include theneurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT)serotypes A-G, as well as those produced by C. baratii and C. butyricum.

Among the clostridial neurotoxins are some of the most potent toxinsknown. By way of example, botulinum neurotoxins have median lethal dose(LD50) values for mice ranging from 0.5 to 5 ng/kg, depending on theserotype. Both tetanus and botulinum toxins act by inhibiting thefunction of affected neurons, specifically the release ofneurotransmitters. While botulinum toxin acts at the neuromuscularjunction and inhibits cholinergic transmission in the peripheral nervoussystem, tetanus toxin acts in the central nervous system.

In nature, clostridial neurotoxins are synthesised as a single-chainpolypeptide that is modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. Cleavage occurs at a specific cleavage site, oftenreferred to as the activation site, that is located between the cysteineresidues that provide the inter-chain disulphide bond. It is thisdi-chain form that is the active form of the toxin. The two chains aretermed the heavy chain (H-chain), which has a molecular mass ofapproximately 100 kDa, and the light chain (L-chain), which has amolecular mass of approximately 50 kDa. The H-chain comprises anN-terminal translocation component (H_(N) domain) and a C-terminaltargeting component (H_(C) domain). The cleavage site is located betweenthe L-chain and the translocation domain components. Following bindingof the HC domain to its target neuron and internalisation of the boundtoxin into the cell via an endosome, the H_(N) domain translocates theL-chain across the endosomal membrane and into the cytosol, and theL-chain provides a protease function (also known as a non-cytotoxicprotease).

Non-cytotoxic proteases act by proteolytically cleaving intracellulartransport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, orSyntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition)John Wiley & Sons, Inc. The acronym SNARE derives from the term SolubleNSF Attachment Receptor, where NSF means N-ethylmaleimide-SensitiveFactor. SNARE proteins are integral to intracellular vesicle fusion, andthus to secretion of molecules via vesicle transport from a cell. Theprotease function is a zinc-dependent endopeptidase activity andexhibits a high substrate specificity for SNARE proteins. Accordingly,once delivered to a desired target cell, the non-cytotoxic protease iscapable of inhibiting cellular secretion from the target cell. TheL-chain proteases of clostridial neurotoxins are non-cytotoxic proteasesthat cleave SNARE proteins.

In view of the ubiquitous nature of SNARE proteins, clostridialneurotoxins such as botulinum toxin have been successfully employed in awide range of therapies.

By way of example, we refer to William J. Lipham, Cosmetic and ClinicalApplications of Botulinum Toxin (Slack, Inc., 2004), which describes theuse of clostridial neurotoxins, such as botulinum neurotoxins (BoNTs),BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and tetanusneurotoxin (TeNT), to inhibit neuronal transmission in a number oftherapeutic and cosmetic or aesthetic applications—for example, marketedbotulinum toxin products are currently approved as therapeutics forindications including focal spasticity, upper limb spasticity, lowerlimb spasticity, cervical dystonia, blepharospasm, hemifacial spasm,hyperhidrosis of the axillae, chronic migraine, neurogenic detrusoroveractivity, glabellar lines, and severe lateral canthal lines. Inaddition, clostridial neurotoxin therapies are described for treatingneuromuscular disorders (see U.S. Pat. No. 6,872,397); for treatinguterine disorders (see US 2004/0175399); for treating ulcers andgastroesophageal reflux disease (see US 2004/0086531); for treatingdystonia (see U.S. Pat. No. 6,319,505); for treating eye disorders (seeUS 2004/0234532); for treating blepharospasm (see US 2004/0151740); fortreating strabismus (see US 2004/0126396); for treating pain (see U.S.Pat. Nos. 6,869,610, 6,641,820, 6,464,986, and U.S. Pat. No. 6,113,915);for treating fibromyalgia (see U.S. Pat. No. 6,623,742, US2004/0062776); for treating lower back pain (see US 2004/0037852); fortreating muscle injuries (see U.S. Pat. No. 6,423,319); for treatingsinus headache (see U.S. Pat. No. 6,838,434); for treating tensionheadache (see U.S. Pat. No. 6,776,992); for treating headache (see U.S.Pat. No. 6,458,365); for reduction of migraine headache pain (see U.S.Pat. No. 5,714,469); for treating cardiovascular diseases (see U.S. Pat.No. 6,767,544); for treating neurological disorders such as Parkinson'sdisease (see U.S. Pat. Nos. 6,620,415, 6,306,403); for treatingneuropsychiatric disorders (see US 2004/0180061, US 2003/0211121); fortreating endocrine disorders (see U.S. Pat. No. 6,827,931); for treatingthyroid disorders (see U.S. Pat. No. 6,740,321); for treatingcholinergic influenced sweat gland disorders (see U.S. Pat. No.6,683,049); for treating diabetes (see U.S. Pat. Nos. 6,337,075,6,416,765); for treating a pancreatic disorder (see U.S. Pat. Nos.6,261,572, 6,143,306); for treating cancers such as bone tumors (seeU.S. Pat. Nos. 6,565,870, 6,368,605, 6,139,845, US 2005/0031648); fortreating otic disorders (see U.S. Pat. Nos. 6,358,926, 6,265,379); fortreating autonomic disorders such as gastrointestinal muscle disordersand other smooth muscle dysfunction (see U.S. Pat. No. 5,437,291); fortreatment of skin lesions associated with cutaneous cell-proliferativedisorders (see U.S. Pat. No. 5,670,484); for management of neurogenicinflammatory disorders (see U.S. Pat. No. 6,063,768); for reducing hairloss and stimulating hair growth (see U.S. Pat. No. 6,299,893); fortreating downturned mouth (see U.S. Pat. No. 6,358,917); for reducingappetite (see US 2004/40253274); for dental therapies and procedures(see US 2004/0115139); for treating neuromuscular disorders andconditions (see US 2002/0010138); for treating various disorders andconditions and associated pain (see US 2004/0013692); for treatingconditions resulting from mucus hypersecretion such as asthma and COPD(see WO 00/10598); and for treating non-neuronal conditions such asinflammation, endocrine conditions, exocrine conditions, immunologicalconditions, cardiovascular conditions, bone conditions (see WO01/21213). All of the above publications are hereby incorporated byreference in their entirety.

The use of non-cytotoxic proteases such as clostridial neurotoxins (e.g.BoNTs and TeNT) in therapeutic and cosmetic treatments of humans andother mammals is anticipated to expand to an ever-widening range ofdiseases and ailments that can benefit from the properties of thesetoxins.

Currently all approved drugs/cosmetic preparations comprising BoNTscontain naturally occurring neurotoxins purified from clostridialstrains (BoNT/A in the case of DYSPORT®, BOTOX® or XEOMIN®, and BoNT/Bin the case of MYOBLOC®).

Recombinant technology offers the possibility of changing or optimizingthe properties of neurotoxins through the introduction of modificationsto its sequence and/or structure. In particular, chimeric neurotoxins inwhich the H_(C) domain or the H_(CC) subdomain is replaced by a H_(C)domain or H_(CC) subdomain from a different neurotoxin have beenproduced.

Rummel et al, 2011 (Exchange of the H_(CC) domain mediating doublereceptor recognition improves the pharmacodynamic properties ofbotulinum neurotoxin. FEBS Journal, 278(23), 4506-4515) generatedvarious active full-length hybrid neurotoxins, including AABB, AACC andBBAA chimera (letters represent the serotype origin of each of the fourdomains: L, H_(N), H_(CN), H_(CC)). The AABB chimera was found to bemore potent than BoNT/A in a mouse phrenic nerve hemidiaphragm assay,while the AACC only retained 10% of the potency of BoNT/A. The BBAAchimera retained 85% of the potency of BoNT/A and was equipotent toBoNT/B.

Wang et al, 2008 (Novel chimeras of botulinum neurotoxins A and E unveilcontributions from the binding, translocation, and protease domains totheir functional characteristics. Journal of Biological Chemistry,283(25), 16993-17002) generated AE (LH_(N) from BoNT/A and H_(C) fromBoNT/E) and EA (LH_(N) from BoNT/E and H_(C) from BoNT/A) chimericneurotoxins, adding a linker in the case of the AE chimera between theLH_(N) and H_(C) domains to increase flexibility. Both were able tocause a paralysis in a mouse phrenic nerve hemidiaphragm assay as wellas in vivo.

Wang et al., 2012a (Longer-acting and highly potent chimaeric inhibitorsof excessive exocytosis created with domains from botulinum neurotoxin Aand B. Biochemical Journal, 444(1), 59-67) generated AB (LH_(N) fromBoNT/A and H_(C) from BoNT/B, with a linker to improve folding) and BA(LH_(N) from BoNT/B and H_(C) from BoNT/A) chimeric neurotoxins. The ABchimera induced a more prolonged neuromuscular paralysis than BoNT/A inmice. The BA chimera was able to reduce exocytosis from non-neuronalcells.

Wang et al, 2012b (Novel chimeras of botulinum and tetanus neurotoxinsyield insights into their distinct sites of neuroparalysis. The FASEBJournal, 26(12), 5035-5048) generated ATx (LH_(N) from BoNT/A and H_(C)from TeNT), TxA (LH_(N) from TeNT and H_(C) from BoNT/A), ETx (LH_(N)from BoNT/E and H_(C) from TeNT) and TxE (LH_(N) from TeNT and H_(C)from BoNT/E) chimera. The information provided with respect to theprotein sequence of these prior art chimeric neurotoxins is summarisedin table 1 below:

TABLE 1 LH_(N) and H_(C) domains in prior art chimeric neurotoxinsChimera LH_(N) H_(C) Rummel, AABB A B: 871-1304 2011 AACC A C: 871-1296BBAA B A: 858-1283 Wang, AE A: 1-874 (+ELGGGGSEL E: 845-1252 2008linker) EA E: 1-844 (+DI linker) A: 871-1296 Wang, AB A: 1-874(+ELGGGGSEL B: 858-1283 2012(a) linker) BA B: 1-861 (+DI linker) A:871-1296 Wang, ATx A: 1-877 Tx: 879-1315 2012(b) TxA Tx: 1-882 (+DIlinker) A: 871-1296 ETx E: 1-844 (+DI linker) Tx: 879-1315 TxE Tx: 1-882(+EL linker) E: 845-1252

However, there still exists a need for an optimized design of chimericneurotoxins allowing for improved therapeutic properties.

The present invention solves the above problem by providing chimericneurotoxins, as specified in the claims.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a chimeric neurotoxin comprising aLH_(N) domain from a first neurotoxin covalently linked to a H_(C)domain from a second neurotoxin, wherein the first and secondneurotoxins are different, wherein the C-terminal amino acid residue ofthe LH_(N) domain corresponds to the first amino acid residue of the 3₁₀helix separating the LH_(N) and H_(C) domains in the first neurotoxin,and wherein the N-terminal amino acid residue of the H_(C) domaincorresponds to the second amino acid residue of the 3₁₀ helix separatingthe LH_(N) and H_(C) domains in the second neurotoxin.

In a second aspect, the invention provides a nucleotide sequenceencoding a chimeric neurotoxin according to the invention.

In a third aspect, the invention provides a vector comprising anucleotide sequence according to the invention.

In a fourth aspect, the invention provides a cell comprising anucleotide sequence or a vector according to the invention.

In a fifth aspect, the invention provides a pharmaceutical compositioncomprising a chimeric neurotoxin according to the invention.

In a sixth aspect, the invention provides a chimeric neurotoxinaccording to the invention for use in therapy.

In a seventh aspect, the invention provides a non-therapeutic use of achimeric neurotoxin according to the invention for treating an aestheticor cosmetic condition.

DETAILED DESCRIPTION

In one aspect, the invention provides a chimeric neurotoxin comprising aLH_(N) domain from a first neurotoxin covalently linked to a H_(C)domain from a second neurotoxin, wherein the first and secondneurotoxins are different,

-   -   wherein the C-terminal amino acid residue of the LH_(N) domain        corresponds to the first amino acid residue of the 3₁₀ helix        separating the LH_(N) and H_(C) domains in the first neurotoxin,        and    -   wherein the N-terminal amino acid residue of the H_(C) domain        corresponds to the second amino acid residue of the 3₁₀ helix        separating the LH_(N) and H_(C) domains in the second        neurotoxin.

As used herein, the term “a”, “an” and “the” can mean one or more.

The term “neurotoxin” as used herein means any polypeptide that enters aneuron and inhibits neurotransmitter release. This process encompassesthe binding of the neurotoxin to a low or high affinity receptor, theinternalisation of the neurotoxin, the translocation of theendopeptidase portion of the neurotoxin into the cytoplasm and theenzymatic modification of the neurotoxin substrate. More specifically,the term “neurotoxin” encompasses any polypeptide produced byClostridium bacteria (clostridial neurotoxins) that enters a neuron andinhibits neurotransmitter release, and such polypeptides produced byrecombinant technologies or chemical techniques. It is this di-chainform that is the active form of the toxin. The two chains are termed theheavy chain (H-chain), which has a molecular mass of approximately 100kDa, and the light chain (L-chain), which has a molecular mass ofapproximately 50 kDa. Preferably, the first and second neurotoxins areclostridial neurotoxins.

An example of a BoNT/A neurotoxin amino acid sequence is provided a SEQID NO: 1 (UNIPROT® accession number A5HZZ9). An example of a BoNT/Bneurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UNIPROT®accession number B1INP5). An example of a BoNT/C neurotoxin amino acidsequence is provided as SEQ ID NO: 3 (UNIPROT® accession number P18640).An example of a BoNT/D neurotoxin amino acid sequence is provided as SEQID NO: 4 (UNIPROT® accession number P19321). An example of a BoNT/Eneurotoxin amino acid sequence is provided as SEQ ID NO: 5 (UNIPROT®accession number Q00496). An example of a BoNT/F neurotoxin amino acidsequence is provided as SEQ ID NO: 6 (UNIPROT® accession number Q57236).An example of a BoNT/G neurotoxin amino acid sequence is provided as SEQID NO: 7 (UNIPROT® accession number Q60393). An example of a TeNTneurotoxin amino acid sequence is provided as SEQ ID NO: 8 (UNIPROT®accession number P04958). The amino acid sequences of said neurotoxinsare shown in the alignment of FIG. 1 below, along with the sequences ofother neurotoxins (i.e. SEQ ID NOs: 58 to 91).

The term “chimeric neurotoxin” as used herein means a neurotoxincomprising or consisting of an LH_(N) domain originating from a firstneurotoxin and a H_(C) domain originating from a second neurotoxin.

The term “H_(C) domain” as used herein means a functionally distinctregion of the neurotoxin heavy chain with a molecular weight ofapproximately 50 kDa that enables the binding of the neurotoxin to areceptor located on the surface of the target cell. The H_(C) domainconsists of two structurally distinct subdomains, the “H_(CN) subdomain”(N-terminal part of the H_(C) domain) and the “H_(CC) subdomain”(C-terminal part of the H_(C) domain), each of which has a molecularweight of approximately 25 kDa.

The term “LH_(N) domain” as used herein means a neurotoxin that isdevoid of the H_(C) domain and consists of an endopeptidase domain (“L”or “light chain”) and the domain responsible for translocation of theendopeptidase into the cytoplasm (H_(N) domain of the heavy chain).

Reference herein to the “first amino acid residue of the 3₁₀ helixseparating the LH_(N) and H_(C) domains in the first neurotoxin” meansthe N-terminal residue of the 3₁₀ helix separating the LH_(N) and H_(C)domains.

Reference herein to the “second amino acid residue of the 3₁₀ helixseparating the LH_(N) and H_(C) domains in the second neurotoxin” meansthe amino acid residue following the N-terminal residue of the 3₁₀ helixseparating the LH_(N) and H_(C) domains.

A “3₁₀ helix” is a type of secondary structure found in proteins andpolypeptides, along with α-helices, β-sheets and reverse turns. Theamino acids in a 3₁₀ helix are arranged in a right-handed helicalstructure where each full turn is completed by three residues and tenatoms that separate the intramolecular hydrogen bond between them. Eachamino acid corresponds to a 120° turn in the helix (i.e., the helix hasthree residues per turn), and a translation of 2.0 Å (=0.2 nm) along thehelical axis, and has 10 atoms in the ring formed by making the hydrogenbond. Most importantly, the N—H group of an amino acid forms a hydrogenbond with the C═O group of the amino acid three residues earlier; thisrepeated i+3→i hydrogen bonding defines a 3₁₀ helix. A 3₁₀ helix is astandard concept in structural biology with which the skilled person isfamiliar.

This 3₁₀ helix corresponds to four residues which form the actual helixand two cap (or transitional) residues, one at each end of these fourresidues. The term “3₁₀ helix separating the LH_(N) and H_(C) domains”as used herein consists of those 6 residues.

Through carrying out structural analyses and sequence alignments, theinventor identified a 3₁₀ helix separating the LH_(N) and H_(C) domainsin tetanus and botulinum neurotoxins. This 3₁₀ helix is surrounded by anα-helix at its N-terminus (i.e. at the C-terminal part of the LH_(N)domain) and by a β-strand at its C-terminus (i.e. at the N-terminal partof the H_(C) domain). The first (N-terminal) residue (cap ortransitional residue) of the 3₁₀ helix also corresponds to theC-terminal residue of this α-helix.

The 3₁₀ helix separating the LH_(N) and H_(C)domains can be for exampledetermined from publically available crystal structures of botulinumneurotoxins, for example 3BTA and 1EPW from RCSB Protein Data Bank forbotulinum neurotoxins A1 and B1 respectively.

In silico modelling and alignment tools which are publically availablecan also be used to determine the location of the 3₁₀ helix separatingthe LH_(N) and H_(C)domains in other neurotoxins, for example thehomology modelling servers LOOPP (Learning, Observing and OutputtingProtein Patterns), PHYRE (Protein Homology/analogY Recognition Engine)and Rosetta, the protein superposition server SuperPose, the alignmentprogram Clustal Omega, and a number of other tools/services listed atthe Internet Resources for Molecular and Cell Biologists. The inventorfound in particular that the region around the “H_(N)/H_(CN)” junctionis structurally highly conserved which renders it an ideal region tosuperimpose different serotypes.

For example, the following methodology was used by the inventor todetermine the sequence of this 3₁₀ helix in other neurotoxins:

-   -   1. The structural homology modelling tool LOOP was used to        obtain a predicted structure of all BoNT serotypes and TeNT        based on the BoNT/A1 crystal structure (3BTA.pdb);    -   2. The structural (pdb) files thus obtained were edited to        include only the N-terminal end of the H_(CN) domain and about        80 residues before it (which are part of the H_(N) domain),        thereby retaining the “H_(N)/H_(CN)” region which is        structurally highly conserved;    -   3. The protein superposition server SuperPose was used to        superpose each serotype onto the 3BTA.pdb structure;    -   4. The superposed pdb files were inspected to locate the 3₁₀        helix at the start of the H_(C) domain of BoNT/A1 and        corresponding residues in the other serotype were then        identified.    -   5. All BoNT serotype sequences were aligned with Clustal Omega        in order to check that corresponding residues were correct.

Examples of LH_(N), H_(C) and 3₁₀ helix domains determined by thismethod are presented in table 2.

TABLE 2 LH_(N), H_(C) and 3₁₀ helix domains SEQ ID Neuro- Accession NOtoxin Number LH_(N) H_(C) 3₁₀ helix (3₁₀ helix) BoNT/A1 A5HZZ9 1-872873-1296 ⁸⁷²NIINTS⁸⁷⁷ 14 BoNT/A2 X73423 1-872 873-1296 ⁸⁷²NIVNTS⁸⁷⁷ 15BoNT/A3 DQ185900 1-872 873-1292 ⁸⁷²NIVNTS⁸⁷⁷ 16 BoNT/A4 EU341307 1-872873-1296 ⁸⁷²NITNAS⁸⁷⁷ 17 BoNT/A5 EU679004 1-872 873-1296 ⁸⁷²NIINTS⁸⁷⁷ 18BoNT/A6 FJ981696 1-872 873-1296 ⁸⁷²NIINTS⁸⁷⁷ 19 BoNT/A7 JQ954969 1-872873-1296 ⁸⁷²NIINTS⁸⁷⁷ 20 BoNT/A8 KM233166 1-872 873-1297 ⁸⁷²NITNTS⁸⁷⁷ 21BoNT/B1 B1INP5 1-859 860-1291 ⁸⁵⁹EILNNI⁸⁶⁴ 22 BoNT/B2 AB084152 1-859860-1291 ⁸⁵⁹EILNNI⁸⁶⁴ 23 BoNT/B3 EF028400 1-859 860-1291 ⁸⁵⁹EILNNI⁸⁶⁴ 24BoNT/B4 EF051570 1-859 860-1291 ⁸⁵⁹EILNNI⁸⁶⁴ 25 BoNT/B5 EF033130 1-859860-1291 ⁸⁵⁹DILNNI⁸⁶⁴ 26 BoNT/B6 AB302852 1-859 860-1291 ⁸⁵⁹EILNNI⁸⁶⁴ 27BoNT/B7 JQ354985 1-859 860-1291 ⁸⁵⁹EILNNI⁸⁶⁴ 28 BoNT/B8 JQ964806 1-859860-1292 ⁸⁵⁹EILNNI⁸⁶⁴ 29 BoNT/C1 P18640 1-867 868-1291 ⁸⁶⁷NINDSK⁸⁷² 30BoNT/CD AB200360 1-867 868-1280 ⁸⁶⁷SINDSK⁸⁷² 31 BoNT/DC AB745660 1-863864-1276 ⁸⁶³SINDSK⁸⁶⁸ 32 BoNT/D P19321 1-863 864-1276 ⁸⁶³SINDSK⁸⁶⁸ 33BoNT/E1 Q00496 1-846 847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 34 BoNT/E2 EF028404 1-846847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 35 BoNT/E3 EF028403 1-846 847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 36BoNT/E4 AB088207 1-846 847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 37 BoNT/E5 AB037711 1-846847-1251 ⁸⁴⁶RIKSSS⁸⁵¹ 38 BoNT/E6 AM695759 1-846 847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 39BoNT/E7 JN695729 1-846 847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 40 BoNT/E8 JN695730 1-846847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 41 BoNT/E9 JX424534 1-846 847-1251 ⁸⁴⁶RIKSSS⁸⁵¹ 42BoNT/E10 KF861917 1-846 847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 43 BoNT/E11 KF861875 1-846847-1252 ⁸⁴⁶RIKSSS⁸⁵¹ 44 BoNT/E12 KM370319 1-846 847-1254 ⁸⁴⁶RIKSSS⁸⁵¹45 BoNT/F1 Q57236 1-865 866-1278 ⁸⁶⁵KIKDNS⁸⁷⁰ 46 BoNT/F2 GU213209 1-865866-1280 ⁸⁶⁵KIKDSS⁸⁷⁰ 47 BoNT/F3 GU213227 1-865 866-1279 ⁸⁶⁵KIKDSS⁸⁷⁰ 48BoNT/F4 GU213214 1-865 866-1277 ⁸⁶⁵KIKDNC⁸⁷⁰ 49 BoNT/F5 GU213211 1-862863-1277 ⁸⁶²KIKDSS⁸⁶⁷ 50 BoNT/F6 M92906 1-864 865-1274 ⁸⁶⁴KIKDSS⁸⁶⁹ 51BoNT/F7 GU213233 1-856 857-1268 ⁸⁵⁶KIKDSS⁸⁶¹ 52 BoNT/G Q60393 1-864865-1297 ⁸⁶⁴NISSNA⁸⁶⁹ 53 BoNT/H KGO15617 1-860 861-1288 ⁸⁶⁰ELKYNC⁸⁶⁵ 54TeNT P04958 1-880 881-1315 ⁸⁸⁰ILKKST⁸⁸⁵ 55

Using structural analysis and sequence alignments, the inventor foundthat the β-strand following the 3₁₀ helix separating the LH_(N) andH_(C) domains is a conserved structure in all botulinum and tetanusneurotoxins and starts at the 8^(th) residue when starting from thefirst residue of the 3₁₀ helix separating the LH_(N) and H_(C) domains(e.g., at residue 879 for BoNT/A1).

According to an alternative definition, the first aspect of theinvention provides a chimeric neurotoxin comprising an LH_(N) domainfrom a first neurotoxin covalently linked to a H_(C) domain from asecond neurotoxin, wherein the first and second neurotoxins aredifferent,

-   -   wherein the C-terminal amino acid residue of the LH_(N) domain        corresponds to the eighth amino acid residue N-terminally to the        β-strand located at the beginning (N-term) of the H_(C) domain        in the first neurotoxin, and    -   wherein the N-terminal amino acid residue of the H_(C) domain        corresponds to the seventh amino acid residue N-terminally to        the β-strand located at the beginning (N-term) of the H_(C)        domain in the second neurotoxin.

According to yet another definition, the first aspect of the inventionprovides a chimeric neurotoxin comprising a LH_(N) domain from a firstneurotoxin covalently linked to a H_(C) domain from a second neurotoxin,wherein the first and second neurotoxins are different,

-   -   wherein the C-terminal amino acid residue of the LH_(N) domain        corresponds to the C-terminal amino acid residue of the α-helix        located at the end (C-term) of LH_(N) domain in the first        neurotoxin, and    -   wherein the N-terminal amino acid residue of the H_(C) domain        corresponds to the amino acid residue immediately C-terminal to        the C-terminal amino acid residue of the α-helix located at the        end (C-term) of LH_(N) domain in the second neurotoxin.

The rationale of the design process of the chimeric neurotoxinsaccording to the invention was to try to ensure that the secondarystructure was not compromised and thereby minimise any changes to thetertiary structure and to the function of each domain.

In some of the prior art chimeric neurotoxins, a linker is requiredbetween the LH_(N) and H_(C) domains (see table 1), presumably to ensureacceptable expression and purification.

Without wishing to be bound by theory, it is hypothesized thatstructuring chimeric neurotoxins in the form of proteins which have atertiary structure closely mimicking the tertiary structure of naturalneurotoxins will facilitate their solubility.

Without wishing to be bound by theory, it is further hypothesized thatthe fact of not disrupting the four central amino acid residues of the3₁₀ helix in the chimeric neurotoxin ensures an optimal conformation forthe chimeric neurotoxin, thereby allowing for the chimeric neurotoxin toexert its functions to their full capacity.

In fact, the inventor has surprisingly found that retaining solely thefirst amino acid residue of the 3₁₀ helix of the first neurotoxin andthe second amino acid residue of the 3₁₀ helix onwards of secondneurotoxin not only allows the production of soluble and functionalchimeric neurotoxins, but further leads to improved properties overother chimeric neurotoxins, in particular an increased potency, anincreased safety ratio and/or a longer duration of action.

Undesired effects of a neurotoxin (caused by diffusion of the neurotoxinaway from the site of administration) can be assessed experimentally bymeasuring percentage bodyweight loss in a relevant animal model (e.g. amouse, where loss of bodyweight is detected within seven days ofadministration). Conversely, desired on-target effects of a neurotoxincan be assessed experimentally by Digital Abduction Score (DAS) assay, ameasurement of muscle paralysis. The DAS assay may be performed byinjection of 20 μL of neurotoxin, formulated in Gelatin PhosphateBuffer, into the mouse gastrocnemius/soleus complex, followed byassessment of Digital Abduction Score using the method of Aoki (Aoki KR,Toxicon 39: 1815-1820; 2001).

In the DAS assay, mice are suspended briefly by the tail in order toelicit a characteristic startle response in which the mouse extends itshind limbs and abducts its hind digits. Following neurotoxin injection,the varying degrees of digit abduction are scored on a five-point scale(0=normal to 4=maximal reduction in digit abduction and leg extension).

The Safety Ratio of a neurotoxin may then be expressed as the ratiobetween the amount of neurotoxin required for a 10% drop in a bodyweightof a mouse (measured at peak effect within the first seven days afterdosing in a mouse) and the amount of neurotoxin required for a DAS scoreof 2. High Safety Ratio scores are therefore desired, and indicate aneurotoxin that is able to effectively paralyse a target muscle withlittle undesired off-target effects.

A high safety ratio is particularly advantageous in therapy because itrepresents an increase in the therapeutic index. In other words, thismeans that reduced dosages can be used compared to known clostridialtoxin therapeutics and/or that increased dosages can be used without anyadditional effects. The possibility to use higher doses of neurotoxinwithout additional effects is particularly advantageous as higher dosesusually lead to a longer duration of action of the neurotoxin.

The Potency of a neurotoxin may be expressed as the minimal dose ofneurotoxin which leads to a given DAS score when administered to a mousegastrocnemius/soleus complex, for example a DAS score of 2 (ED₅₀ dose)or a DAS score of 4. The Potency of a neurotoxin may be also expressedas the EC₅₀ dose in a cellular assay measuring SNARE cleavage by theneurotoxin, for example the EC₅₀ dose in a cellular assay measuringSNAP-25 cleavage by a chimeric BoNT/AB neurotoxin.

The duration of action of a neurotoxin may be expressed as the timerequired for retrieving a DAS score of 0 after administration of a givendose of neurotoxin, for example the minimal dose of neurotoxin leadingto a DAS score of 4, to a mouse gastrocnemius/soleus complex.

In one embodiment, the first neurotoxin is a Botulinum Neurotoxin (BoNT)serotype A, serotype B, serotype C, serotype D, serotype E, serotype For serotype G or a Tetanus Neurotoxin (TeNT), and the second neurotoxinis a Botulinum Neurotoxin (BoNT) serotype A, serotype B, serotype C,serotype D, serotype E, serotype F or serotype G or a Tetanus Neurotoxin(TeNT). In a preferred embodiment, the first neurotoxin is a BotulinumNeurotoxin (BoNT) serotype A, serotype B or a serotype C, and the secondneurotoxin is a Botulinum Neurotoxin (BoNT) serotype A, serotype B or aserotype C.

Different BoNT serotypes can be distinguished based on inactivation byspecific neutralising anti-sera, with such classification by serotypecorrelating with percentage sequence identity at the amino acid level.BoNT proteins of a given serotype are further divided into differentsubtypes on the basis of amino acid percentage sequence identity.

Preferably, the first and second neurotoxins are Botulinum Neurotoxinsfrom different serotypes. In another embodiment, either the first orsecond neurotoxin is a Botulinum Neurotoxin and the other neurotoxin isa Tetanus neurotoxin.

Using an “XY” representation according to which X is the LH_(N) domainand Y is the H_(C) domain, the following chimeric neurotoxins areembodiments of the present invention:

-   -   AB, AC, AD, AE, AF, AG, ATx,    -   BA, BC, BD, BE, BF, BG, BTx,    -   CA, CB, CD, CE, CF, CG, CTx,    -   DA, DB, DC, DE, DF, DG, DTx,    -   EA, EB, EC, ED, EF, EG, ETx,    -   FA, FB, FC, FD, FE, FG, FTx,    -   GA, GB, GC, GD, GE, GF, FTx,    -   TxA, TxB, TxC, TxD, TxE, TxF, TxG,

wherein A, B, C, D, E, F, G and Tx are respectively Botulinum Neurotoxin(BoNT) serotype A, serotype B, serotype C, serotype D, serotype E,serotype F, serotype G and Tetanus Neurotoxin (TeNT).

Yet, using the same “XY” representation as described above, thefollowing chimeric neurotoxins are preferred embodiments of the presentinvention:

-   -   AB, AC,    -   BA, BC,    -   CA, CB,

wherein A, B, C, are respectively Botulinum Neurotoxin (BoNT) serotypeA, serotype B, and serotype C.

In one embodiment, the LH_(N) domain from the first neurotoxincorresponds to:

-   -   amino acid residues 1 to 872 of SEQ ID NO: 1, or a polypeptide        sequence having at least 70% sequence identity thereto,    -   amino acid residues 1 to 859 of SEQ ID NO: 2, or a polypeptide        sequence having at least 70% sequence identity thereto,    -   amino acid residues 1 to 867 of SEQ ID NO: 3, or a polypeptide        sequence having at least 70% sequence identity thereto,    -   amino acid residues 1 to 863 of SEQ ID NO: 4, or a polypeptide        sequence having at least 70% sequence identity thereto,    -   amino acid residues 1 to 846 of SEQ ID NO: 5, or a polypeptide        sequence having at least 70% sequence identity thereto,    -   amino acid residues 1 to 865 of SEQ ID NO: 6, or a polypeptide        sequence having at least 70% sequence identity thereto,    -   amino acid residues 1 to 864 of SEQ ID NO: 7, or a polypeptide        sequence having at least 70% sequence identity thereto G, or    -   amino acid residues 1 to 880 of SEQ ID NO: 8, or a polypeptide        sequence having at least 70% sequence identity thereto.

and the H_(C) domain from the second neurotoxin corresponds to:

-   -   amino acid residues 873 to 1296 of SEQ ID NO: 1, or a        polypeptide sequence having at least 70% sequence identity        thereto,    -   amino acid residues 860 to 1291 of SEQ ID NO: 2, or a        polypeptide sequence having at least 70% sequence identity        thereto,    -   amino acid residues 868 to 1291 of SEQ ID NO: 3, or a        polypeptide sequence having at least 70% sequence identity        thereto,    -   amino acid residues 864 to 1276 of SEQ ID NO: 4, or a        polypeptide sequence having at least 70% sequence identity        thereto,    -   amino acid residues 847 to 1251 of SEQ ID NO: 5, or a        polypeptide sequence having at least 70% sequence identity        thereto,    -   amino acid residues 866 to 1275 of SEQ ID NO: 6, or a        polypeptide sequence having at least 70% sequence identity        thereto,    -   amino acid residues 865 to 1297 of SEQ ID NO: 7, or a        polypeptide sequence having at least 70% sequence identity        thereto, or    -   amino acid residues 881 to 1315 of SEQ ID NO: 8, or a        polypeptide sequence having at least 70% sequence identity        thereto.

The “percent sequence identity” between two or more nucleic acid oramino acid sequences is a function of the number of identicalnucleotides/amino acids at identical positions shared by the alignedsequences. Thus, % identity may be calculated as the number of identicalnucleotides/amino acids at each position in an alignment divided by thetotal number of nucleotides/amino acids in the aligned sequence,multiplied by 100. Calculations of % sequence identity may also takeinto account the number of gaps, and the length of each gap that needsto be introduced to optimize alignment of two or more sequences.Sequence comparisons and the determination of percent identity betweentwo or more sequences can be carried out using specific mathematicalalgorithms, in particular a global alignment mathematical algorithm(Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972) such asBLAST, which will be familiar to a skilled person.

The first or second neurotoxin can be a mosaic neurotoxin. The term“mosaic neurotoxin” as used in this context refers to a naturallyoccurring clostridial neurotoxin that comprises at least one functionaldomain from another type of clostridial neurotoxins (e.g. a clostridialneurotoxin of a different serotype), said clostridial neurotoxin notusually comprising said at least one functional domain. Examples ofmosaic neurotoxins are naturally occurring BoNT/DC and BoNT/CD. BoNT/DCcomprises the L chain and H_(N) domain of serotype D and the H_(C)domain of serotype C, whereas BoNT/CD consists of the L chain and H_(N)domain of serotype C and the H_(C) domain of serotype D.

The first and second neurotoxins can be modified neurotoxins andderivatives thereof, including but not limited to those described below.A modified neurotoxin or derivative may contain one or more amino acidsthat has been modified as compared to the native (unmodified) form ofthe neurotoxin, or may contain one or more inserted amino acids that arenot present in the native (unmodified) form of the toxin. By way ofexample, a modified clostridial neurotoxin may have modified amino acidsequences in one or more domains relative to the native (unmodified)clostridial neurotoxin sequence. Such modifications may modifyfunctional aspects of the neurotoxin, for example biological activity orpersistence. Thus, in one embodiment, the first neurotoxin and/or thesecond neurotoxin is a modified neurotoxin, or modified neurotoxinderivative.

A modified neurotoxin retains at least one of the functions of aneurotoxin, selected from the ability to bind to a low or high affinityneurotoxin receptor on a target cell, to translocate the endopeptidaseportion of the neurotoxin (light chain) into the cell cytoplasm and tocleave a SNARE protein. Preferably, a modified neurotoxin retains atleast two of these functions. More preferably a modified neurotoxinretains these three functions.

A modified neurotoxin may have one or more modifications in the aminoacid sequence of the heavy chain (such as a modified H_(C) domain),wherein said modified heavy chain binds to target nerve cells with ahigher or lower affinity than the native (unmodified) neurotoxin. Suchmodifications in the H_(C) domain can include modifying residues in theganglioside binding site of the H_(C) domain or in the protein (SV2 orsynaptotagmin) binding site that alter binding to the gangliosidereceptor and/or the protein receptor of the target nerve cell. Examplesof such modified neurotoxins are described in WO 2006/027207 and WO2006/114308, both of which are hereby incorporated by reference in theirentirety.

A modified neurotoxin may have one or more modifications in the aminoacid sequence of the light chain, for example modifications in thesubstrate binding or catalytic domain which may alter or modify theSNARE protein specificity of the modified LC. Examples of such modifiedneurotoxins are described in WO 2010/120766 and US 2011/0318385, both ofwhich are hereby incorporated by reference in their entirety.

A modified neurotoxin may comprise one or more modifications thatincreases or decreases the biological activity and/or the biologicalpersistence of the modified neurotoxin. For example, a modifiedneurotoxin may comprise a leucine- or tyrosine-based motif, wherein saidmotif increases or decreases the biological activity and/or thebiological persistence of the modified neurotoxin. Suitableleucine-based motifs include xDxxxLL, xExxxLL, xExxxlL, and xExxxLM(wherein x is any amino acid). Suitable tyrosine-based motifs includeY-x-x-Hy (wherein Hy is a hydrophobic amino acid). Examples of modifiedneurotoxins comprising leucine- and tyrosine-based motifs are describedin WO 2002/08268, which is hereby incorporated by reference in itsentirety.

In one embodiment, the first or second neurotoxin is a modified BoNT/Awhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1.

In one embodiment, the first or second neurotoxin is a modified BoNT/Bwhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2.

In one embodiment, the first or second neurotoxin is a modified BoNT/Cwhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 3.

In one embodiment, the first or second neurotoxin is a modified BoNT/Dwhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4.

In one embodiment, the first or second neurotoxin is a modified BoNT/Ewhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5.

In one embodiment, the first or second neurotoxin is a modified BoNT/Fwhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 6.

In one embodiment, the first or second neurotoxin is a modified BoNT/Gwhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 7.

In one embodiment, the first or second neurotoxin is a modified TeNTwhich has an amino acid sequence having at least 70%, preferably atleast 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 8.

In one embodiment, the second neurotoxin is a BoNT/B. Such a chimericneurotoxin is referred to herein as a “BoNT/XB neurotoxin”.

In a preferred embodiment, the first neurotoxin is a BoNT/A and thesecond neurotoxin is a BoNT/B. Such a chimeric neurotoxin is referred toherein as a “BoNT/AB neurotoxin”. More preferably, the first neurotoxinis a BoNT/A1 and the second neurotoxin is a BoNT/B1. More preferablystill, the LH_(N) domain from a first neurotoxin corresponds to aminoacid residues 1 to 872 of BoNT/A1 and the H_(C) domain from a secondneurotoxin corresponds to amino acid residues 860 to 1291 of BoNT/B1. Inone preferred embodiment, the LH_(N) domain from a first neurotoxincorresponds to amino acid residues 1 to 872 of SEQ ID NO: 1 and theH_(C) domain from a second neurotoxin corresponds to amino acid residues860 to 1291 of SEQ ID NO: 2. In other words, a preferred chimericneurotoxin according to the invention comprises or consists of the aminoacid sequence SEQ ID NO:13.

Compared to the BoNT/A serotype, natural BoNT/B is much less potentdespite a comparatively greater abundance of its receptor on synapticvesicles. This is due to a unique amino acid change within the toxinbinding site in human synaptotagmin II (Syt II) as compared to rodent(rat/mouse) Syt II (Peng, L., et al., J Cell Sci, 125(Pt 13):3233-42(2012); Rummel, A. et al, FEBS J 278:4506-4515 (2011).13,22. As a resultof this residue change, human Syt II has greatly diminished binding tonatural BoNT/B, as well as to natural BoNT/D-C, and/G. These findingsprovide an explanation for the clinical observations that a much higherdose of BoNT/B than BoNT/A (which binds a different receptor) is neededto achieve the same levels of therapeutic effects in patients. In apreferred embodiment of a BoNT/XB or BoNT/AB neurotoxin according to theinvention, the H_(C) domain from a BoNT/B neurotoxin comprises at leastone amino acid residue substitution, addition or deletion in the H_(CC)subdomain which has the effect of increasing the binding affinity ofBoNT/B neurotoxin for human Syt II as compared to the natural BoNT/Bsequence.

Suitable amino acid residue substitution, addition or deletion in theBoNT/B H_(CC) subdomain have been disclosed in WO2013/180799 and inPCT/US2016/024211 which is not yet published (both herein incorporatedby reference).

Suitable amino acid residue substitution, addition or deletion in theBoNT/B H_(CC) subdomain include substitution mutations selected from thegroup consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T;S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W,S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P andcombinations thereof.

Suitable amino acid residue substitution, addition or deletion in theBoNT/B H_(CC) subdomain further include combinations of two substitutionmutations selected from the group consisting of: E1191M and S1199L,E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q andS1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191Cand S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W,E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

Suitable amino acid residue substitution, addition or deletion in theBoNT/B H_(CC) subdomain also include a combination of three substitutionmutations which are E1191M, S1199W and W1178Q.

In a preferred embodiment, the suitable amino acid residue substitution,addition or deletion in the BoNT/B H_(CC) subdomain include acombination of two substitution mutations which are E1191M and S1199Y.In other words, a preferred chimeric neurotoxin according to theinvention comprises or consists of the amino acid sequence SEQ ID NO: 11or SEQ ID NO: 12.

In another preferred embodiment, the first neurotoxin is a BoNT/C andthe second neurotoxin is a BoNT/B. Such a chimeric neurotoxin isreferred to herein as a “BoNT/CB neurotoxin”. More preferably, the firstneurotoxin is a BoNT/C1 and the second neurotoxin is a BoNT/B1. Morepreferably still, the LH_(N) domain from a first neurotoxin correspondsto amino acid residues 1 to 867 of BoNT/C1 and the H_(C) domain from asecond neurotoxin corresponds to amino acid residues 860 to 1291 ofBoNT/B1. In one preferred embodiment, the LH_(N) domain from a firstneurotoxin corresponds to amino acid residues 1 to 867 of SEQ ID NO: 3and the H_(C) domain from a second neurotoxin corresponds to amino acidresidues 860 to 1291 of SEQ ID NO: 2. In a preferred embodiment, theH_(C) domain from the BoNT/B neurotoxin comprises at least one aminoacid residue substitution, addition or deletion in the H_(CC) subdomainwhich has the effect of increasing the binding affinity of BoNT/Bneurotoxin for human Syt II as compared to the natural BoNT/B sequence.Suitable amino acid residue substitution, addition or deletion in theBoNT/B H_(CC) subdomain is as described above.

In a preferred embodiment of a BoNT/XDC neurotoxin (chimeric neurotoxinin which the second neurotoxin is a mosaic BoNT/DC) according to theinvention, the H_(C) domain from a mosaic BoNT/DC neurotoxin comprisesat least one amino acid residue substitution, addition or deletion inthe H_(CC) subdomain which has the effect of increasing the bindingaffinity of mosaic BoNT/DC neurotoxin for human Syt II as compared tothe natural mosaic BoNT/DC sequence.

In a preferred embodiment of a BoNT/XG neurotoxin according to theinvention, the H_(C) domain from a BoNT/G neurotoxin comprises at leastone amino acid residue substitution, addition or deletion in the H_(CC)subdomain which has the effect of increasing the binding affinity ofBoNT/G neurotoxin for human Syt II as compared to the natural BoNT/Gsequence.

Other preferred neurotoxins according to the invention are as follows.

In a preferred embodiment, the first neurotoxin is a BoNT/A and thesecond neurotoxin is a BoNT/C. Such a chimeric neurotoxin is referred toherein as a “BoNT/AC neurotoxin”. More preferably, the first neurotoxinis a BoNT/A1 and the second neurotoxin is a BoNT/C1. More preferablystill, the LH_(N) domain from a first neurotoxin corresponds to aminoacid residues 1 to 872 of BoNT/A1 and the H_(C) domain from a secondneurotoxin corresponds to amino acid residues 868 to 1291 of BoNT/C1. Inone preferred embodiment, the LH_(N) domain from a first neurotoxincorresponds to amino acid residues 1 to 872 of SEQ ID NO: 1 and theH_(C) domain from a second neurotoxin corresponds to amino acid residues868 to 1291 of SEQ ID NO: 3.

In another preferred embodiment, the first neurotoxin is a BoNT/B andthe second neurotoxin is a BoNT/A. Such a chimeric neurotoxin isreferred to herein as a “BoNT/BA neurotoxin”. More preferably, the firstneurotoxin is a BoNT/B1 and the second neurotoxin is a BoNT/A1. Morepreferably still, the LH_(N) domain from a first neurotoxin correspondsto amino acid residues 1 to 859 of BoNT/B1 and the H_(C) domain from asecond neurotoxin corresponds to amino acid residues 873 to 1296 ofBoNT/A1. In one preferred embodiment, the LH_(N) domain from a firstneurotoxin corresponds to amino acid residues 1 to 859 of SEQ ID NO: 2and the H_(C) domain from a second neurotoxin corresponds to amino acidresidues 873 to 1293 of SEQ ID NO: 1.

In another preferred embodiment, the first neurotoxin is a BoNT/B andthe second neurotoxin is a BoNT/C. Such a chimeric neurotoxin isreferred to herein as a “BoNT/BC neurotoxin”. More preferably, the firstneurotoxin is a BoNT/B1 and the second neurotoxin is a BoNT/C1. Morepreferably still, the LH_(N) domain from a first neurotoxin correspondsto amino acid residues 1 to 859 of BoNT/B1 and the H_(C) domain from asecond neurotoxin corresponds to amino acid residues 868 to 1291 ofBoNT/C1. In one preferred embodiment, the LH_(N) domain from a firstneurotoxin corresponds to amino acid residues 1 to 859 of SEQ ID NO: 2and the H_(C) domain from a second neurotoxin corresponds to amino acidresidues 868 to 1291 of SEQ ID NO: 3. In other words, a preferredchimeric neurotoxin according to the invention comprises or consists ofthe amino acid sequence SEQ ID NO: 56.

In another preferred embodiment, the first neurotoxin is a BoNT/C andthe second neurotoxin is a BoNT/A. Such a chimeric neurotoxin isreferred to herein as a “BoNT/CA neurotoxin”. More preferably, the firstneurotoxin is a BoNT/C1 and the second neurotoxin is a BoNT/A1. Morepreferably still, the LH_(N) domain from a first neurotoxin correspondsto amino acid residues 1 to 867 of BoNT/C1 and the H_(C) domain from asecond neurotoxin corresponds to amino acid residues 873 to 1296 ofBoNT/A1. In one preferred embodiment, the LH_(N) domain from a firstneurotoxin corresponds to amino acid residues 1 to 867 of SEQ ID NO: 3and the H_(C) domain from a second neurotoxin corresponds to amino acidresidues 873 to 1296 of SEQ ID NO: 1.

The chimeric neurotoxins of the present invention can be produced usingrecombinant technologies. Thus, in one embodiment, a chimeric neurotoxinaccording to the invention is a recombinant chimeric neurotoxin. Itshall be readily understood that, according to this preferredembodiment, a nucleotide sequence encoding a recombinant chimericneurotoxin of the invention, a vector comprising said nucleotidesequence, and a cell comprising said vector, as further described below,can mutatis mutandis be referred as being recombinant.

In another aspect, the invention provides a nucleotide sequence encodinga chimeric neurotoxin according to the invention, for example a DNA orRNA sequence. In a preferred embodiment, the nucleotide sequence is aDNA sequence.

The nucleic acid molecules of the invention may be made using anysuitable process known in the art. Thus, the nucleic acid molecules maybe made using chemical synthesis techniques. Alternatively, the nucleicacid molecules of the invention may be made using molecular biologytechniques.

The DNA sequence of the present invention is preferably designed insilico, and then synthesised by conventional DNA synthesis techniques.

The above-mentioned nucleic acid sequence information is optionallymodified for codon-biasing according to the ultimate host cell (e.g. E.coli) expression system that is to be employed.

In another aspect, the invention provides a vector comprising anucleotide sequence according to the invention. In one embodiment, thenucleic acid sequence is prepared as part of a DNA vector comprising apromoter and terminator. In a preferred embodiment, the vector has apromoter selected from Tac, AraBAD, T7-Lac, or T5-Lac.

A vector may be suitable for in vitro and/or in vivo expression of theabove-mentioned nucleic acid sequence. The vector can be a vector fortransient and/or stable gene expression. The vector may additionallycomprise regulatory elements and/or selection markers. Said vector maybe of viral origin, of phage origin, or of bacterial origin. Forexample, said expression vector may be a pET, pJ401, pGEX vector or aderivative thereof.

In another aspect, the invention provides a cell comprising a nucleotidesequence or a vector according to the invention. The term “cell” canherein be used interchangeably with the term “host cell” or “cell line”.Suitable cell type includes prokaryotic cells, for example E. coli, andeukaryotic cells, such as yeast cells, mammalian cells, insect cells,etc. Preferably, the cell is E. coli.

In another aspect, the invention provides a method for producing achimeric neurotoxin according to the invention, said method comprisingthe step of culturing a cell as described above, under conditionswherein said chimeric neurotoxin is produced. Said conditions arewell-known to the skilled practitioner and therefore need not be furtherdetailed herein. Preferably, said method further comprises the step ofrecovering the chimeric neurotoxin from the culture.

In another aspect, the invention provides a pharmaceutical compositioncomprising a chimeric neurotoxin according to the invention. Preferably,the pharmaceutical composition comprises a chimeric neurotoxin togetherwith at least one component selected from a pharmaceutically acceptablecarrier, excipient, adjuvant, propellant and/or salt.

In another aspect, the invention provides a chimeric neurotoxin orpharmaceutical composition according to the invention for use intherapy. More precisely, the invention relates to the use of a chimericneurotoxin or pharmaceutical composition as described herein, formanufacturing a medicament. In other words, the invention relates to amethod for treating a subject in need thereof, comprising the step ofadministering an effective amount of a chimeric neurotoxin orpharmaceutical composition as described herein, to said subject. By“effective amount”, it is meant that the chimeric neurotoxin orpharmaceutical composition is administered in a quantity sufficient toprovide the effect for which it is indicated. As used herein, the term“subject” preferably refers to a human being or an animal, morepreferably to a human being.

A chimeric neurotoxin according to the invention is preferably suitablefor use in treating a condition associated with unwanted neuronalactivity in a subject in need thereof, for example a condition selectedfrom the group consisting of spasmodic dysphonia, spasmodic torticollis,laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervicaldystonia, focal hand dystonia, blepharospasm, strabismus, hemifacialspasm, eyelid disorder, cerebral palsy, focal spasticity and other voicedisorders, spasmodic colitis, neurogenic bladder, anismus, limbspasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagiaand other muscle tone disorders and other disorders characterized byinvoluntary movements of muscle groups, lacrimation, hyperhidrosis,excessive salivation, excessive gastrointestinal secretions, secretorydisorders, pain from muscle spasms, headache pain, migraine anddermatological conditions. More precisely, the invention relates to theuse of a chimeric neurotoxin or pharmaceutical composition as describedherein, for manufacturing a medicament intended to treat a conditionassociated with unwanted neuronal activity, as described above. In otherwords, the invention relates to a method for treating a conditionassociated with unwanted neuronal activity, as described above, in asubject in need thereof, said method comprising the step ofadministering an effective amount of a chimeric neurotoxin orpharmaceutical composition as described herein, to said subject.

In another aspect, the invention provides a non-therapeutic use of achimeric neurotoxin according to the invention for treating an aestheticor cosmetic condition, in a subject in need thereof. In other words, theinvention relates to a method for treating an aesthetic or cosmeticcondition in a subject in need thereof, comprising the step ofadministering an effective amount of a chimeric neurotoxin orpharmaceutical composition as described herein, to said subject.According to this aspect of the invention, the subject to be treated ispreferably not suffering from a condition associated with unwantedneuronal activity as described above. More preferably, said subject is ahealthy subject, i.e. a subject which is not suffering from any disease.

In another aspect, the invention provides a kit for use in a therapeuticor non-therapeutic (cosmetic or aesthetic) method, or for a therapeuticor non-therapeutic (cosmetic or aesthetic) use, as described above, saidkit comprising a pharmaceutical composition of the invention andinstructions for performing said method or use. More precisely, theinvention relates to a kit comprising a pharmaceutical composition ofthe invention and instructions for therapeutic or cosmeticadministration of said composition to a subject in need thereof. As usedherein, the term “instructions” refers to a publication, a recording, adiagram, or any other medium of expression which can be used tocommunicate how to perform a method or use of the invention, such astherapeutic or cosmetic administration of said composition to a subjectin need thereof. Said instructions can, for example, be affixed to acontainer which comprises said composition or said kit.

The engineered chimeric neurotoxins of the present invention may beformulated for oral, parenteral, continuous infusion, inhalation ortopical application. Compositions suitable for injection may be in theform of solutions, suspensions or emulsions, or dry powders which aredissolved or suspended in a suitable vehicle prior to use.

In the case of a chimeric neurotoxin that is to be delivered locally,the chimeric neurotoxin may be formulated as a cream (e.g. for topicalapplication), or for sub-dermal injection.

Local delivery means may include an aerosol, or other spray (e.g. anebuliser). In this regard, an aerosol formulation of a chimericneurotoxin enables delivery to the lungs and/or other nasal and/orbronchial or airway passages.

Chimeric neurotoxins of the invention may be administered to a patientby intrathecal or epidural injection in the spinal column at the levelof the spinal segment involved in the innervation of an affected organ.

A preferred route of administration is via laparoscopic and/orlocalised, particularly intramuscular, injection.

The dosage ranges for administration of the chimeric neurotoxins of thepresent invention are those to produce the desired therapeutic effect.It will be appreciated that the dosage range required depends on theprecise nature of the chimeric neurotoxin or composition, the route ofadministration, the nature of the formulation, the age of the patient,the nature, extent or severity of the patient's condition,contraindications, if any, and the judgement of the attending physician.Variations in these dosage levels can be adjusted using standardempirical routines for optimisation.

Fluid dosage forms are typically prepared utilising the chimericneurotoxin and a pyrogen-free sterile vehicle. The engineeredclostridial toxin, depending on the vehicle and concentration used, canbe either dissolved or suspended in the vehicle. In preparing solutionsthe chimeric neurotoxin can be dissolved in the vehicle, the solutionbeing made isotonic if necessary by addition of sodium chloride andsterilised by filtration through a sterile filter using aseptictechniques before filling into suitable sterile vials or ampoules andsealing. Alternatively, if solution stability is adequate, the solutionin its sealed containers may be sterilised by autoclaving.Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents and orlocal anaesthetic agents may be dissolved in the vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicleprior to use, may be prepared by filling pre-sterilised ingredients intoa sterile container using aseptic technique in a sterile area.Alternatively, the ingredients may be dissolved into suitable containersusing aseptic technique in a sterile area. The product is then freezedried and the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Administration in accordance with the present invention may takeadvantage of a variety of delivery technologies including microparticleencapsulation, viral delivery systems or high-pressure aerosolimpingement.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, any nucleic acidsequences are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aclostridial neurotoxin” includes a plurality of such candidate agentsand reference to “the clostridial neurotoxin” includes reference to oneor more clostridial neurotoxins and equivalents thereof known to thoseskilled in the art, and so forth.

The invention will now be described, by way of example only, withreference to the following Figures and Examples.

DESCRIPTION OF THE DRAWINGS

FIG. 1—Sequence alignment of BoNT/A1-8, /B1-8, /C, /D, /E1-12, /F1-7,/G, /“H”, and TeNT using CLUSTAL Omega (1.2.1) multiple sequencealignment tool. The location of the putative 3₁₀ helix separating theLH_(N) and H_(C) domains is in bold and underlined characters.

FIG. 2—SDS PAGE of purified recombinant BoNT/AB chimera 1, 2 and 3A (SEQID NO: 9, 10 and 11 respectively). Lanes are labelled “Marker”(molecular weight marker), “−DTT” (oxidised BoNT/AB chimera sample), and“+DTT” (reduced BoNT/AB chimera sample).

FIG. 3—Cleavage of SNAP-25 in rat spinal cord neurons by recombinantBoNT/AB chimera 1, 2 and 3A (SEQ ID Nos:9, 10 and 11 respectively).Cultured rat primary spinal cord neurons (SCN) were exposed to variousconcentrations of recombinant BoNT/AB chimera 1, 2 or 3A for 24 hours,at 37° C. in a humidified atmosphere with 10% C(CO₂. Cells were thenlysed with 1× NUPAGE™ buffer supplemented with DTT and BENZONASE®nuclease. The samples were transferred to microcentrifuge tubes, heatedfor 5 min at 90° C. on heat block and stored at −20° C. before analysisof SNAP-25 cleavage by Western blot. SNAP-25 was detected using apolyclonal antibody, that detects both the full length and cleaved formsof SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) Vas used asthe secondary antibody.

FIG. 4—Mouse digit abduction scoring assay. Mice were injected into thegastrocnemius-soleus complex muscles of one hind limb, under shortgeneral anaesthesia; muscle weakening was measured on a 0-4 scale usingthe digit abduction score (DAS). DAS max values were determined for eachdose and plotted against dose and the data were fitted to a 4-parameterlogistic equation, ED₅₀ and dose leading to DAS 4 (DAS 4 dose) valueswere determined.

FIG. 5—SDS PAGE of purified recombinant BoNT/AB chimera 3B and 3C (SEQID NO: 12 and 13 respectively). Lanes are labelled “Marker” (molecularweight marker), “−DTT” (oxidised BoNT/AB chimera sample), and “+DTT”(reduced BoNT/AB chimera sample).

FIG. 6—Cleavage of SNAP-25 by recombinant BoNT/A and BoNT/AB chimera 3Band 3C (SEQ ID NOs: 1, 12 and 13 respectively) in human inducedpluripotent stem cell derived peripheral neurons (PERL4U-Axiogenesis,Germany). PERL4U cells were exposed to various concentrations ofrecombinant BoNT/A, or BoNT/AB chimera 3B or 3C for 24 hours, at 37° C.in a humidified CO₂ atmosphere containing 5% CO₂. Cells were then lysedwith 1× NUPAGE™ buffer supplemented with DTT and BENZONASE® nuclease.The samples were transferred to microcentrifuge tubes, heated for 5 mainat 90° C. on heat block and stored at −20° C., before analysis ofSNAP-25 cleavage by Western blot. SNAP-25 was detected using apolyclonal antibody, that detects both the full length and cleaved formsof SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used asthe secondary antibody.

FIG. 7—Duration of muscle weakening over time in the mouse digitabduction scoring assay. Mice were injected into thegastrocnemius-soleus complex muscles of one hind limb, under shortgeneral anaesthesia; muscle weakening was measured on a 0-4 scale usingthe digit abduction score (DAS). Animals of the group injected with thelowest dose that induced during the first four days of injection a DASof 4 were monitored until complete recovery of the muscle weakness to aDAS of 0 (no observed muscle weakness).

FIG. 8—SDS PAGE of purified recombinant BoNT/BC (SEQ ID NO: 56). Lanesare labelled “Marker” (molecular weight marker), “−DTT” (oxidisedBoNT/BC chimera sample), and “+DTT” (reduced BoNT/BC chimera sample).

FIG. 9—Cleavage of VAMP-2 by native BoNT/B, BoNT/BC chimera, andinactive recombinant BoNT/B (SEQ ID NOs: 56 and 57, respectively) in ratcortical neurons. Cells were exposed to various concentrations of BoNTfor 24 hours, at 37° C. in a humidified CO₂ atmosphere containing 5%CO₂. Cells were lysed with 1× NUPAGE™ buffer supplemented with DTT andBENZONASE® nuclease, and heated for 5 min at 90° C. before storage at−20° C. Samples were analysed for VAMP-2 cleavage by Western blot usinga polyclonal rabbit anti-VAMP-2 (Abeam ab3347, 1:1000) primary antibody,and an HRP-conjugated anti-rabbit secondary antibody (Sigma #A6154).

FIG. 10—Cleavage of VAMP-2 peptide reporter by native BoNT/B and BoNT/BCchimera (SEQ ID NO: 2 and 56, respectively) using the BOTEST® BoNTdetection kit (BioSentinel). Various concentrations of BoNT wereincubated at 30° C. for 18 hours with a VAMP-2 peptide with a CFP-YFPFRET pair as a reporter and the proportion of uncleaved:cleaved reportersubstrate was measured as a loss of YFP fluorescence intensity at 528 nmand gain of CFP fluorescence at 485 nm following excitation at 440 nm.

EXAMPLES

The following Examples serve to illustrate particular embodiments of theinvention, and do not limit the scope of the invention defined in theclaims in any way.

Example 1 Mapping of 3₁₀ Helix in Clostridial Neurotoxins

The amino acid sequences of all BoNT serotypes and TeNT were obtainedfrom a public database (e.g., UNIPROT® or the National Center forBiotechnology Information) NCBI and then modelled onto the known crystalstructure of BoNT/A1 (3BTA.pdb) using LOOPP. This yielded a predictedprotein structure which was edited to retain only to N-terminal part ofthe HCN domain and ^(˜)80 residues before it (C-terminal part of theH_(N) domain)—this domain (“H_(N)/H_(CN)”) is structurally highlyconserved, therefore, making it the best region to superimpose differentserotypes. Each edited structure was then superimposed onto 3BTA.pdbusing SuperPose and the residues corresponding to a conspicuous 3₁₀helixat the start of the H_(C) of BoNT/A1 (⁸⁷² NIINTS⁸⁷⁶) and correspondingresidues in the other serotype were then identified. These werecross-checked with a sequence alignment of all BoNT serotypes withClustal Omega (FIG. 1).

By identifying this region of structural equivalence between differentneurotoxins, it was possible to identify a specific point at which aC-terminal half of one neurotoxin may transition over to a N-terminalhalf of another neurotoxin without interrupting the secondary structureof the overall molecule. This point was chosen to be the start of the3₁₀ helix.

The results are presented in table 2 above.

Example 2 Cloning, Expression and Purification of BoNT/AB Chimeras

BoNT/AB chimeric constructs 1, 2, 3A, 3B, and 3C (SEQ ID NO: 9 to 13)were constructed from DNA encoding the parent serotype molecule andappropriate oligonucleotides using standard molecular biologytechniques. These were then cloned into the pJ401 expression vector withor without a C-terminal His₁₀-tag and transformed into BLR (DE3) E. colicells for over-expression. These cells were grown at 37° C. and 225 RPMshaking in 2 L baffled conical flasks containing 1 L modified TerrificBroth (mTB) supplemented with the appropriate antibiotic. Once the A₆₀₀reached >0.5, the incubator temperature was decreased to 16° C., andthen induced with 1 mM IPTG an hour later for 20 h at 225 RPM shaking,to express the recombinant BoNT/AB construct.

Harvested cells were lysed by ultrasonication and clarified bycentrifugation at 4500 RPM for 1 h at 4° C. The recombinant BoNT/ABchimeric molecules were then extracted in ammonium sulphate and purifiedby standard fast protein liquid chromatography (FPLC) techniques. Thisinvolved using a hydrophobic interaction resin for capture and ananion-exchange resin for the intermediate purification step. Thepartially purified molecules were then proteolytically cleaved withendoproteinase Lys-C to yield the active di-chain. This was furtherpurified with a second hydrophobic interaction resin to obtain the finalBoNT/AB chimera.

For BoNT/AB chimeric molecules with a decahistadine tag (H₁₀) (chimera1, 2, 3A), the capture step employed the use of an immobilised nickelresin instead of the hydrophobic interaction resin.

The sequence of each chimera is presented in table 3.

TABLE 3 chimeric BoNT/AB constructs Molecule SEQ ID NO Sequence Chimera1 9 A1: 1-871 + B1: 858-1291 (E1191M/S1199Y) + His₁₀-tag Chimera 2 10A1: 1-874 + ELGGGGSEL + B1: 858-1291 (E1191M/S1199Y) + His₁₀-tag Chimera3A 11 A1: 1-872 + B1: 860-1291 (E1191M/S1199Y) + His₁₀-tag Chimera 3B 12A1: 1-872 + B1: 860-1291 (E1191M/S1199Y) Chimera 3C 13 A1: 1-872 + B1:860-1291

Example 3 Comparison of BoNT/AB Chimera 1, 2 and 3A

BoNT/AB chimera 1, 2 and 3A which have a C-terminal His₁₀ tag andE1191M/S1199Y double mutation were purified as described in Example 1(FIG. 2) and tested for functional activity.

Rat Spinal Cord Neurons SNAP-25 Cleavage Assay

Primary cultures of rat spinal cord neurons (SCN) were prepared andgrown, for 3 weeks, in 96 well tissue culture plates (as described in:Masuyer et al., 2011, J. Struct. Biol. Structure and activity of afunctional derivative of Clostridium botulinum neurotoxin B; and in:Chaddock et al., 2002, Protein Expr. Purif. Expression and purificationof catalytically active, non-toxic endopeptidase derivatives ofClostridium botulinum toxin type A). Serial dilutions of BoNT/AB wereprepared in SCN feeding medium. The growth medium from the wells to betreated was collected and filtered (0.2 μm filter). 125 μL of thefiltered medium was added back to each test well. 125 μL of dilutedtoxin was then added to the plate (triplicate wells). The treated cellswere incubated at 37° C., 10% CO₂, for 24±1 h).

Analysis of BoNT Activity Using the SNAP-25 Cleavage Assay

Following treatment, BoNT was removed and cells were washed once in PBS(Gibco, UK). Cells were lysed in 1× NUPAGE™ lysis buffer (LifeTechnologies) supplemented with 0.1 M dithiothreitol (DTT) and 250units/mL BENZONASE® nuclease (Sigma). Lysate proteins were separated bySDS-PAGE and transferred to nitrocellulose membranes. Membranes wereprobed with a primary antibody specific for SNAP-25 (Sigma #S9684) whichrecognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by the BoNT/Aendopeptidase. The secondary antibody used was an HRP-conjugatedanti-rabbit IgG (Sigma #A6154). Bands were detected by enhancedchemiluminescence and imaged using a pXi6 Access (Synoptics, UK). Theintensity of bands was determined using GENETOOLS® software (Syngene,Cambridge, UK) and the percentage of SNAP-25 cleaved at eachconcentration of BoNT calculated. Data were fitted to a 4-parameterlogistic equation and pEC₅₀ calculated using GRAPHPAD PRISM® version 6(GraphPad).

Table 4 below provides the pEC₅₀ values determined for Chimera 1, 2 and3A in the rat SCN SNAP-25 cleavage assay. These results show that thethree BoNT/AB chimeras retained the ability to enter rat spinal cordneurons and cleave their target substrate. However, chimera 3A was morepotent than chimera 1 and 2 in this assay (see also FIG. 3).

TABLE 4 pEC₅₀ ± SEM Chimera 1 12.42 ± 0.04 Chimera 2 12.57 ± 0.01Chimera 3A 12.89 ± 0.04

Digit Abduction Scoring (DAS) Assay

The method to measure the activity of BoNT/AB chimera 1, 2 and 3A in theDAS assay is based on the startled response toe spreading reflex ofmice, when suspended briefly by the tail. This reflex is scored as DigitAbduction Score (DAS) and is inhibited after administration of BoNT intothe gastrocnemius-soleus muscles of the hind paw. Mice are suspendedbriefly by the tail to elicit a characteristic startled response inwhich the animal extends its hind limb and abducts its hind digits.(Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10)

On the day of injection, mice were anaesthetized in an induction chamberreceiving isoflurane 3% in oxygen. Each mouse received an intramuscularinjection of BoNT/AB chimera or vehicle (phosphate buffer containing0.2% gelatine) in the gastrocnemius-soleus muscles of the right hindpaw.

Following neurotoxin injection, the varying degrees of digit abductionwere scored on a scale from zero to four, where 0=normal and 4=maximalreduction in digit abduction and leg extension. ED₅₀ was determined bynonlinear adjustment analysis using average of maximal effect at eachdose. The mathematical model used was the 4 parameters logistic model.

DAS was performed every 2 hours during the first day after dosing;thereafter it was performed 3 times a day for 4 days.

FIG. 4 shows the fitted curves for chimera 1, 2 and 3A (SEQ ID NO: 9, 10and 11 respectively). The chimera 3A curve is shifted to the left,meaning lower doses of chimera 3A achieved a similar DAS responsecompared to chimera 1 and 2, therefore showing that chimera 3A is morepotent than the others in the mouse DAS assay; see also the table below(table 5) that provides the values for the calculated ED₅₀ and the doseleading to DAS 4 (highest score) for each chimera.

Table 5 below provides the ED₅₀ and DAS 4 doses determined forrecombinant BoNT/A1 (rBoNT/A1) and chimeras 1, 2 and 3A in the mouse DASassay. These results show that of the three chimeras, chimera 3A has thehighest in vivo potency in inducing muscle weakening. Studies shown inFIG. 4 and table 5 were performed in mice obtained from Charles Riverlaboratories.

TABLE 5 ED₅₀ DAS 4 dose (pg/mouse) (pg/mouse) rBoNT/A1 1 5 Chimera 1 23200 Chimera 2 89 >300 Chimera 3A 18 133

Example 4 Comparison of BoNT/AB Chimera 3B, 3C and BoNT/A1

Untagged BoNT/AB chimera 3B and 3C, respectively with and without thepresence of the E1191M/S1199Y double mutation (SEQ ID NO: 12 and 13)were purified as described in Example 1 (FIG. 5), and tested forfunctional activity using recombinant BoNT/A1 (SEQ ID NO: 1) as areference.

Human Pluripotent Stem Cells SNAP-25 Cleavage Assay

Cryopreserved PERI.4U-cells were purchased from Axiogenesis (Cologne,Germany). Thawing and plating of the cells were performed as recommendedby the manufacturer. Briefly, cryovials containing the cells were thawedin a water bath at 37° C. for 2 minutes. After gentle resuspension thecells were transferred to a 50 mL tube. The cryovial was washed with 1mL of PERI.4U® thawing medium supplied by the manufacturer and themedium was transferred drop-wise to the cell suspension to the 50 mLtube, prior to adding a further 2 mL of PERI.4U® thawing mediumdrop-wise to the 50 mL tube. Cells were then counted using ahemocytometer. After this, a further 6 mL of PERI.4U® thawing medium wasadded to the cell suspension. A cell pellet was obtained bycentrifugation at 260 xg (e.g. 1,100 RPM) for 6 minutes at roomtemperature. Cells were then resuspended in complete PERI.4U® culturemedium supplied by the manufacturer. Cells were plated at a density of50,000 to 150,000 cells per cm² on cell culture plates coated withpoly-L-ornithine and laminin. Cells were cultured at 37° C. in ahumidified CO₂ atmosphere, and medium was changed completely every 2-3days during culture.

For toxin treatment, serial dilutions of BoNTs were prepared in PERI.4U®culture medium. The medium from the wells to be treated was collectedand filtered (0.2 μm filter). 125 μL of the filtered medium was addedback to each test well. 125 μL of diluted toxin was then added to theplate (triplicate wells). The treated cells were incubated at 37° C.,10% CO₂, for 48±1 h).

Analysis of BoNT Activity Using the SNAP-25 Cleavage Assay

Following treatment, BoNT was removed and cells were washed once in PBS(Gibco, UK). Cells were lysed in 1× NUPAGE™ lysis buffer (LifeTechnologies) supplemented with 0.1 M dithiothreitol (DTT) and 250units/mL BENZONASE® nuclease (Sigma). Lysate proteins were separated bySDS-PAGE and transferred to nitrocellulose membranes. Membranes wereprobed with a primary antibody specific for SNAP-25 (Sigma #S9684) whichrecognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by the BoNT/Aendopeptidase. The secondary antibody used was an HRP-conjugatedanti-rabbit IgG (Sigma #A6154). Bands were detected by enhancedchemiluminescence and imaged using a pXi6 Access (Synoptics, UK). Theintensity of bands was determined using GENETOOLS® software (Syngene,Cambridge, UK) and the percentage of SNAP-25 cleaved at eachconcentration of BoNT calculated. Data were fitted to a 4-parameterlogistic equation and pEC₅₀ calculated using GRAPHPAD PRISM® version 6(GraphPad).

FIG. 6 shows that chimera 3B and 3C displayed greater potency thanrBoNT/A1 in cleaving SNAP-25 in induced human pluripotent stem cells butthe former significantly more so. This can be explained by the doublemutation which increases the affinity of chimera 3B for the humansynaptotagmin II protein receptor present in these cells (FIG. 6, table6).

TABLE 6 pEC₅₀ ± SEM rBoNT/A1 10.21 ± 0.05 Chimera 3B 12.38 ± 0.06Chimera 3C 10.72 ± 0.08

Digit Abduction Scoring (DAS) Assay—Safety Ratio

The method to measure the activity of BoNTs in the DAS assay is based onthe startled response toe spreading reflex of mice, when suspendedbriefly by the tail. This reflex is scored as Digit Abduction Score(DAS) and is inhibited after administration of BoNT into thegastrocnemius-soleus muscles of the hind paw. Mice are suspended brieflyby the tail to elicit a characteristic startled response in which theanimal extends its hind limb and abducts its hind digits. (Aoki et al.1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10)

On the day of injection, mice were anaesthetized in an induction chamberreceiving isoflurane 3% in oxygen. Each mouse received an intramuscularinjection of BoNT or vehicle (phosphate buffer containing 0.2% gelatine)in the gastrocnemius-soleus muscles of the right hind paw.

Following neurotoxin injection, the varying degrees of digit abductionwere scored on a scale from zero to four, where 0=normal and 4=maximalreduction in digit abduction and leg extension. ED₅₀ was determined bynonlinear adjustment analysis using average of maximal effect at eachdose. The mathematical model used was the 4 parameters logistic model.

DAS was performed every 2 hours during the first day after dosing;thereafter it was performed 3 times a day for 4 days for all doses.Animals of the groups injected with vehicle and the lowest dose thatinduced during the first four days of injection a DAS of 4 werethereafter monitored until complete recovery of the muscle weakness to aDAS of 0 (no observed muscle weakness).

For calculation of the safety ratio all animals were weighed the daybefore toxin injection (D0) and thereafter once daily throughout theduration of the study. The average body weight, its standard deviation,and the standard error mean were calculated daily for each dose-group.To obtain the safety ratio for a BoNT (−10%ΔBW/ED₅₀), the dose at whichat any time during the study the average weight of a dose-group waslower than 10% of the average weight at D0 of that same dose-group wasdivided by the ED₅₀ for the BoNT studied. The lethal dose was defined asthe dose at which one or more of the animals within that dose-groupdied.

FIG. 7 shows the duration of muscle weakening over time in the mousedigit abduction scoring assay for rBoNT/A1, chimera 3B and chimera 3C(SEQ ID NO: 1, 12 and 13), showing that the chimera has longer durationof action.

Table 7 below provides the ED₅₀ and DAS 4 doses determined for rBoNT/A1and chimeras 3B and 3C in the mouse DAS assay. The table also providethe total duration of action for the DAS 4 dose until complete recoveryof the muscle weakness to a DAS of 0 (no observed muscle weakness). Inaddition, the table shows the mouse lethal dose and the safety ratio(−10%, ΔBW/ED₅₀), as defined in the text above. In comparison torBoNT/A1, chimeras 3B and 3C have longer duration of action, a bettersafety ratio, and a higher lethal dose. Studies shown in FIG. 7 andtable 7 were performed in mice obtained from Janvier laboratories.

TABLE 7 Total ED₅₀ duration (DAS 2) DAS 4 of action Mouse Safety Dosedose (day) with lethal ratio (pg/ (pg/ lowest DAS dose (−10% mouse)mouse) 4 dose (pg) ΔBW/ED₅₀) rBoNT/A1 0.9 2.3 29 18 4.5 Chimera 3B 8.089 42 200 14.1 Chimera 3C 5.0 26 42 8.9 7.4

Example 5 Expression and Purification of BoNT/BC Chimera andConfirmation of Functional Activity

BoNT/BC chimera 4 (SEQ ID NO: 56) was cloned, expressed and purified asdescribed in Example 2 except for the use of a different expression cellstrain (BL21) and proteolytic cleavage with trypsin rather thanendoproteinase Lys-C (FIG. 8).

TABLE 8 chimeric BoNT/BC construct Molecule SEQ ID NO Sequence Chimera 456 B1: 1-859 + C1: 868-1291

This chimera was tested for functional activity in a VAMP-2 cleavageassay.

Rat Cortical Neuron VAMP-2 Cleavage Assay

Rat cortical neurons were prepared and maintained on poly-L-ornithine(PLO) coated 96-well plates at a density of 20000 cells/well in 125 μLNEUROBASAL® media containing 2% B27 supplement, 0.5 mM GLUTAMAX™supplement, 1% foetal bovine serum (FBS) and 100 U/mLpenicillin/streptomycin, at 37° C. in a humidified atmosphere containing5% CO₂. A further 125 μL NEUROBASAL® medium containing 2% B27, 0.5 mMGLUTAMAX™ supplement was added on DIV 4. Cells were maintained byreplacement of half the medium every 3-4 days. On DIV 11, 1.5 μMcytosine β-D-arabinofuranoside (AraC) was added to the medium to preventproliferation of non-neuronal cells. Cortical neurons at DIV 19-21 weretreated with a concentration range of BoNT (30 fM-3 nM) for 24 hours at37° C.

Analysis of BoNT Activity Using the VAMP-2 Cleavage Assay

Cells were briefly washed in assay medium (NEUROBASAL® media w/o phenolred, 2% B27, 0.5 mM GLUTAMAX™ supplement, 10 μM TFB-TBOA((3S)-3-[[3-[[4-(Trifluoromethyl) benzoyl] amino] phenyl]methoxy]-L-aspartic acid, before lysis in 100 μL lysis buffer (NUPAGE®LDS sample buffer, 1 mM DTT and 1:500 BENZONASE® nuclease) and heated at90° C. for 5 minutes. 15 μL lysates were run in 12% Bis-Tris gels at 200V for 50 minutes with MES buffer. Proteins were transferred ontonitrocellulose membranes via a TRANS-BLOT® TURBO™ transfer system(Biorad) using the low MW program Membranes were blocked with 5% low fatmilk in PBST and then probed with rabbit anti-VAMP-2 (Abcam ab3347,1:1000) primary antibody and then HRP-conjugated anti-rabbit secondaryantibody (Sigma #A6154). Membranes were developed with SUPERSIGNAL® WestDura chemiluminescent substrate and visualized using a SYNGENE® PXisystem. Band densitometry was analyzed using GENETOOLS® software(Syngene) and VAMP-2 percentage cleavage at each concentration of BoNTwas determined relative to the control wells. Data was fit to a4-parameter logistic equation and the pEC₅₀ calculated using GRAPHPADPRISM® software (GraphPad).

FIG. 9 shows that chimera 4 is able to bind to rat spinal cord neurones,translocate into the cytoplasm, and specifically cleave its substrateVAMP-2. As a point of reference, this chimera is clearly functionalcompared an inactive recombinant BoNT/B1 molecule (having a doublemutation at E231Q and H234Y, and also referred herein as BoNT/B1(0))(SEQ ID NO: 57), and is almost as active as the native BoNT/B1 molecule(SEQ ID NO: 2) (Table 9). This may be explained by the high affinitybinding of BoNT/B to synaptotagmin and various gangliosides present onthe rat cell surface, whereas the binding domain of C in chimera 4, isonly known to bind with lower affinity to gangliosides. This issupported by data from the light chain protease activity assay, as shownfurther below.

TABLE 9 pEC50 ± SEM (VAMP-2 cleavage assay in rat cortical cells) nativeBoNT/B1 10.60 ± 0.06  Chimera 4 9.36 ± 0.15 BoNT/B1(0) inactive

Light Chain Protease Activity Assay

The light chain activity of serotype B was assessed using the BOTEST®(BioSentinel A1009) cell-free assay according to the manufacturer'sinstructions. For example, BoNTs were diluted to 1.39 nM in BOTEST®Reaction Buffer (50 mM HEPES-NaOH, 5 mM NaCl, 10 μM ZnCl₂, 0.1%Tween-20, 0.1 mg/ml BSA, pH 7.1) and reduced with 5 mM DTT for 30minutes at room temperature. VAMP-2 peptide reporter (CFP-VAMP-2(33-94)-YFP in 50 mM HEPES-NaOH, 10 mM NaCl, 15% glycerol) at a finalconcentration of 200 nM was combined with a concentration range of BoNTs(500 fM-1.25 nM, final) in black MAXISORP® plates (Nunc) in a finalassay volume of 100 μL/well. The plates were sealed and incubated at 30°C. for 18 hours away from light. The loss of CFP to YFP FRETfluorescence at 528 nm and gain of GFP fluorescence at 485 nm followingexcitation at 440 nm was measured using a BIOTEK® SYNGERY® HT platereader. The fluorescence emission ratio of uncleaved:cleaved reportersubstrate at each BoNT concentration was fit to a 4-parameter logisticequation and the pEC₅₀ calculated using GRAPHPAD PRISM® software.

The light chain protease activity assay confirms that the light chain ofchimera 4 is as active as the one of native BoNT/B1 (see FIG. 10, andTable 10), and therefore that, as explained above, the results of theVAMP-2 cleavage assay may be explained by the fact that BoNT/B has ahigher affinity to synaptotagmin and various gangliosides present on therat cell surface, compared to the binding domain of C in chimera 4 whichonly binds to gangliosides.

TABLE 10 pEC50 ± SEM (VAMP-2 peptide cleavage assay in vitro) nativeBoNT/B1 10.62 ± 0.01 Chimera 4 10.46 ± 0.01 BoNT/B1(0) NA

The invention claimed is:
 1. A chimeric neurotoxin comprising a LH_(N) domain from a first neurotoxin covalently linked to a H_(C) domain from a second neurotoxin, wherein: the first and second neurotoxins are different; the C-terminal amino acid residue of the LH_(N) domain corresponds to the first amino acid residue of the 3₁₀ helix separating the LH_(N) and H_(C) domains in the first neurotoxin; and the N-terminal amino acid residue of the H_(C) domain corresponds to the second amino acid residue of the 3₁₀ helix separating the LH_(N) and H_(C) domains in the second neurotoxin.
 2. The chimeric neurotoxin of claim 1, wherein the first and second neurotoxins are each individually selected from botulinum neurotoxin (BoNT) serotypes A, B, C, D, E, F, or G, or Tetanus neurotoxin (TeNT).
 3. The chimeric neurotoxin of claim 1, wherein the LH_(N) domain corresponds to: amino acid residues 1 to 872 of SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto; amino acid residues 1 to 859 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto; amino acid residues 1 to 867 of SEQ ID NO: 3, or a sequence having at least 70% sequence identity thereto; amino acid residues 1 to 863 of SEQ ID NO: 4, or a sequence having at least 70% sequence identity thereto; amino acid residues 1 to 846 of SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto; amino acid residues 1 to 865 of SEQ ID NO: 6, or a sequence having at least 70% sequence identity thereto; amino acid residues 1 to 864 of SEQ ID NO: 7, or a sequence having at least 70% sequence identity thereto; or amino acid residues 1 to 880 of SEQ ID NO: 8, or a sequence having at least 70% sequence identity thereto; and wherein the H_(C) domain corresponds to: amino acid residues 873 to 1296 of SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto; amino acid residues 860 to 1291 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto; amino acid residues 868 to 1291 of SEQ ID NO: 3, or a sequence having at least 70% sequence identity thereto; amino acid residues 864 to 1276 of SEQ ID NO: 4, or a sequence having at least 70% sequence identity thereto; amino acid residues 847 to 1251 of SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto; amino acid residues 866 to 1275 of SEQ ID NO: 6, or a sequence having at least 70% sequence identity thereto; amino acid residues 865 to 1297 of SEQ ID NO: 7, or a sequence having at least 70% sequence identity thereto; or amino acid residues 881 to 1315 of SEQ ID NO: 8, or a sequence having at least 70% sequence identity thereto.
 4. The chimeric neurotoxin of claim 1, wherein the first neurotoxin is a BoNT/A and wherein the second neurotoxin is a BoNT/B.
 5. The chimeric neurotoxin of claim 4, wherein the first neurotoxin is a BoNT/A1 and the second neurotoxin is a BoNT/B1.
 6. The chimeric neurotoxin of claim 5, wherein the LH_(N) domain corresponds to amino acid residues 1 to 872 of SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto, and the H_(C) domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto.
 7. The chimeric neurotoxin of claim 4, wherein the H_(C) domain comprises at least one amino acid residue substitution, addition or deletion in the H_(CC) subdomain that has the effect of increasing the binding affinity of the H_(C) domain for the human Syt II receptor as compared to the natural sequence.
 8. The chimeric neurotoxin of claim 7, wherein the H_(C) domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and comprises an amino acid substitution selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C; E1191V, E1191L; E1191Y; S1199W; S1199E; S1199H; W1178Y; W1178Q; W1178A; W1178S; Y1183C; and Y1183P.
 9. The chimeric neurotoxin of claim 7, wherein the H_(C) domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and comprises two amino acid substitutions selected from the group consisting of: E1191M and S1199L; E1191M and S1199Y; E1191M and S1199F; E1191Q and S1199L; E1191Q and S1199Y; E1191Q and S1199F; E1191M and S1199W; E1191M and W1178Q; E1191C and S1199W; E1191C and S1199Y; E1191C and W1178Q; E1191Q and S1199W; E1191V and S1199W; E1191V and S1199Y; and E1191V and W1178Q.
 10. The chimeric neurotoxin of claim 9, wherein the substitutions are E1191M and S1199Y.
 11. The chimeric neurotoxin of claim 7, wherein the H_(C) domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and comprises the following substitutions: E1191M; S1199W; and W1178Q.
 12. The chimeric neurotoxin of claim 1, wherein the first neurotoxin is a BoNT/B and the second neurotoxin is a BoNT/C.
 13. The chimeric neurotoxin of claim 12, wherein the first neurotoxin is a BoNT/B1 and the second neurotoxin is a BoNT/C1.
 14. The chimeric neurotoxin of claim 13, wherein the LH_(N) domain corresponds to amino acid residues 1 to 859 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and the H_(C) domain corresponds to amino acid residues 868 to 1291 of SEQ ID NO: 3, or a sequence having at least 70% sequence identity thereto.
 15. A pharmaceutical composition comprising the chimeric neurotoxin of claim 1, and a pharmaceutically acceptable carrier, an excipient, an adjuvant, a propellant, and/or a salt.
 16. A kit comprising the pharmaceutical composition of claim 15 and instructions for therapeutic or cosmetic administration of the composition to a subject in need thereof.
 17. A method for producing the chimeric neurotoxin of claim 1, the method comprising the step of culturing a cell comprising a nucleotide sequence encoding the chimeric neurotoxin under conditions wherein the chimeric neurotoxin is produced. 